Methods for Isolating Monocytes

ABSTRACT

The present invention provides HIDE1 as novel monocyte markers. Since HIDE1 are membrane proteins, monocytes can be specifically detected by using antibodies that bind to HIDE1. Further, HIDE1-positive monocytes can also be collected from peripheral blood or the like using a cell sorter, magnet, or such. Monocytes that can be prepared based on the present invention are useful in cell immunotherapy.

This application is a U.S. National Phase Application, filed under 35U.S.C. §371 of Patent Cooperation Treaty Application No.PCT/JP2005/00923, filed Jan. 25, 2005 and claims the benefit of JapanesePatent Application Number 2004-018747, filed Jan. 27, 2004, the contentsof each of the aforementioned applications are hereby incorporated byreference in their entirety.

TECHNICAL FIELD

The present invention relates to antibodies for detecting monocytemarkers, and uses thereof.

BACKGROUND ART

Monocytes are cells which migrate in the blood and which havephagocytotic activity, belonging to the group of mononuclear phagocytes.Once differentiated, monocytes remain in the bone marrow for only shorttime before entering the circulatory system, where they remain forseveral days. Monocytes then infiltrate tissues and body cavities, anddifferentiate into macrophages and dendritic cells. Monocytes are knownto increase in the circulatory system in inflammatory diseases,post-transplantation rejection reactions, infectious disease recoveryphases, monocytic leukemia, and such.

Immunotherapy is a therapeutic method aiming to potentiate theimmunological function of patients using substances that activate immunecells, cytokines, antibodies, and the like. Immunotherapy is being notedas an auxiliary therapy to improve the therapeutic effect of othertreatments or to prevent recurrences and the like. A representativeimmunotherapy includes cell immunotherapy. Typically, cell immunotherapycomprises the steps of collecting immune cells from a patient; growingand activating the cells in vitro; and then returning the cells to thepatient. Such cells to be used in cell immunotherapy can include, forexample, lymphocytes and dendritic cells.

One antitumor immunotherapy using lymphocytes is a known method whichuses peripheral blood lymphocytes, tumor tissue-infiltratinglymphocytes, and the like, which are activated or grown using cytokines(lymphokine-activated killer (LAK) cell therapy; J. Immunol. (1984) 132:2123-8; J. Exp. Med. (1982) 155: 1823-41). In LAK therapy, for example,lymphocytes are separated from a patient's blood; a cytokine such asinterleukin-2 (IL-2) is added to those peripheral blood lymphocytes thathave not received antigen stimulation, and lymphokine-activated killercells (LAK cells) with strong antitumor activity are prepared; and thenthe LAK cells are returned to the patient.

From the 1990s great strides have been made in the study of dendriticcells (DCs). DCs are known as cells with strong antigen-presentingactivity. Since DCs are the most efficient cells in terms of presentingantigens and activating T lymphocytes, their application to cellimmunotherapy has been proposed. Methods of cell immunotherapy usingdendritic cells are, for example, ex vivo ingestion of tumor cellantigens (cancer specific antigens) by a patient's dendritic cells topotentiate cancer antigen-presenting ability; and then the return ofthese cells to the patient to induce an immune response against thepatient's cancer. When dendritic cells that have phagocytosed antigensex vivo are administered to patients, they are thought to induce helperT cells in vivo, and these helper T cells activate killer T cells andnatural killer cells, thereby potentiating the patient's ability toeliminate the cancer.

The first clinical application of this kind of cell immunotherapy usingdendritic cells targeted melanomas. In immune responses that take placeupon invasion of a foreign antigen into the body, dendritic cells thathave phagocytosed a foreign antigen migrate to the lymph nodes andpresent the antigen to lymphocytes. Thus, therapeutic methods comprisingthe injection of dendritic cells directly into lesions such as tumorsare also being used (dendritic cell injection (DCI)). Also in practicaluse are methods that comprise mixing dendritic cells and activatedlymphocytes to activate both cells, and then administering these cellsto patients (DCAT therapy).

Examples of other therapeutic methods using blood cells includemacrophage-based therapies for spinal cord damage. There are reportsthat axon regrowth is induced by direct administration of macrophages tothe lesion site of rats whose spinal cord has been cut. Methods fortreating human paralysis caused by spinal cord damage, which compriseinjecting activated autologous macrophages into spinal cord parenchymaltissues, are also being assessed from a clinical viewpoint.

Many secretory and membrane proteins are known to have receptorfunctions, and to transmit cellular signals that initiate variouscellular responses upon ligand binding. In general, many ligandsinvolved in such cellular responses are also pharmaceutically important.Thus, many studies are being performed to identify ligands andreceptors. Patent Documents 1 and 2 also aim to identify this kind ofreceptor, and describe the preparation of cDNAs encoding thetransmembrane protein called “human TANGO353” from among clones in amixed lymphocyte reaction library. Based only on its origin, PatentDocuments 1 and 2 state that TANGO353 can be used to proliferate,differentiate, and activate T or B cells, and to relieve symptomsinvolved in the functional abnormality of these cells, and so on.However, Patent Documents 1 and 2 do not make more detailedinvestigation of the protein's distribution of expression, actualfunction, and the like, and thus the relationship between TANGO353 andmonocytes is unclear from the descriptions of these Documents.

At present, an enormous volume of genome sequence information has beendisclosed. Gene expression profiling is thought to be greatly useful inelucidating biological events involving genes whose sequences have beendetermined. High coverage expression profiling (HiCEP) technology is amethod developed based on amplified fragment length polymorphism (AFLP;Vos et al., Nucleic Acids Res. (1995) 23: 4407-17). Expression profilingusing HiCEP technology does not require sequence information and candetect non-coding transcripts, and known and unidentified genes.

[Patent Document 1] U.S. Patent Application NO. 2002/0055139

[Patent Document 2] WO 01/09162

[Non-patent Document 1] Fukumura et al., Nucleic Acids Res. (2003) 31:e94

DISCLOSURE OF THE INVENTION

Dendritic cells and macrophages to be used in cell immunotherapy or suchcan be induced to differentiate from monocytes. Thus, methods usingautologous peripheral blood monocytes separated from patients are inwide clinical use. In methods using peripheral blood monocytes, themonocytes are first separated by apheresis or density centrifugation.Such physical methods require repeated centrifugation, and areproblematic in that they significantly damage cells and further increasethe chance of bacterial contamination. Some surface antigens, includingCD14, CD11b, and CD33, are known as monocyte markers; however, morespecific monocyte markers are required. Thus, an objective of thepresent invention is to identify proteins to become markers that arehighly expressed in monocytes.

The mRNAs of about 60,000 and 57,000 genes were respectively collectedfrom immature and mature dendritic cells of mouse spleens. Differencesin expression levels between the respective genes were determined usingHiCEP technology (Non-patent Document 1). As a result, “High expressionGene of Immature Dendritic cells 1 (HIDE1)”, a single transmembraneprotein gene expressed at high levels in immature dendritic cells, wasidentified (SEQ ID NO: 1; the amino acid sequence encoded by the gene isshown in SEQ ID NO: 2). Meanwhile, a splice variant that did notcomprise a transmembrane domain was recovered during PCR cloning of thefull-length gene. This suggests the existence of a secretory HIDE1(soluble HIDE1: sHIDE1) (nucleotide sequence: SEQ ID NO: 3; amino acidsequence: SEQ ID NO: 4). The amino acid sequences of membrane HIDE1 andsecretory HIDE1 are compared in FIG. 1.

HIDE1 was expressed at high levels in murine immature dendritic cells,and other researchers had already disclosed a human homolog of HIDE1 ina database (XM_(—)172995). The nucleotide sequence of human HIDE1 andthe amino acid sequence encoded by the same are shown in SEQ ID NOs: 5and 6, respectively (FIGS. 2 and 3). The nucleotide sequence homologyand amino acid sequence homology between mouse and human HIDE1 werefound to be 73.9% and 68.4%, respectively.

The human HIDE1 shown by the present invention to be a novel monocytemarker has a sequence 100% identical to that of the TANGO353 describedin Patent Documents 1 and 2. However, Patent Documents 1 and 2 describethe cloning of TANGO353 from a mixture comprising T and B lymphocytes,and only suggest the possibility that TANGO353 is involved in thedifferentiation and growth of T and B lymphocytes based on its origin.Specifically, these documents neither describe nor suggest theexpression of TANGO353 in monocytes, nor its use as a monocyte marker.

Human HIDE1 gene was cloned from a placental cDNA library, and that geneproduct was used to immunize mice, producing a monoclonal antibody(anti-HIDE1 antibody) that can be used in FCM, WB, andimmunoprecipitation (FIG. 4). Then, human peripheral blood mononuclearcells (PBMCs) were stained with the anti-HIDE1 antibody, and the resultsshowed that HIDE1 was expressed mainly in monocytes, but not inlymphocytes. Slight expression was also observed in granulocytes, butwith much weaker staining than the monocytes (FIG. 5). Furthermore,double staining was carried out using the anti-HIDE1 antibody andanother monocyte marker (CD14, CD11b, or CD33). The results showed thatthe anti-HIDE1 antibody stained monocytes more broadly than CD14, andmonocytes could be stained more specifically when using HIDE1 as amarker than when using CD11b.

Furthermore, HIDE1 expression was found in human monocytic cultured celllines such as HL60, U937, and THP-1. It was thus revealed that HIDE1could be used as a marker for monocytes, including such cultured celllines. When THP-1, a monocytic cultured cell line, was differentiatedinto macrophage-like cells using phorbol ester as adifferentiation-inducing factor, HIDEl expression markedly decreased(FIG. 7 d). This result strongly suggests that HIDE1 is expressedspecifically in monocytes, and emphasizes that HIDE1 can be used as amonocyte marker.

As described above, the present invention demonstrated that HIDE1 couldbe used as a specific monocyte marker. Monocytes developed anddifferentiated from bone marrow stem cells in vivo are known to migratefrom blood into tissues, and then differentiate into dendritic cells(DCs) and macrophages with more activated functions. Thus, not only canmonocytes be detected and isolated by screening cells using the HIDE1monocyte markers of the present invention as an indicator, but DCs andmacrophages can also be prepared by differentiating the isolatedmonocytes in vitro using known techniques. Further, since HIDE1 is notexpressed in lymphocytes, HIDE1 can also be used as a tool to enrichlymphocytes by removing HIDE1-positive cells from peripheral blood usingan anti-HIDE1antibody. Thus, the present invention provides thefollowing antibodies and uses thereof:

-   [1] an antibody for detecting a monocyte marker, which binds to a    protein or a polypeptide selected from the group of:    -   (1) an HIDE1 protein,    -   (2) a protein encoded by a nucleotide sequence that hybridizes        to a complementary sequence of an HIDE1 gene under stringent        conditions, and    -   (3) a polypeptide fragment with at least eight amino acid        residues, wherein the fragment is derived from the protein of        the above (1) or (2);-   [2] a method for detecting a monocyte, which comprises the steps of:    -   (1) contacting the antibody of [1] with a blood cell sample        predicted to comprise a monocyte, and    -   (2) detecting a blood cell that has bound to the antibody in        step (1);-   [3] a method for isolating a monocyte, which comprises the steps of:    -   (1) contacting the antibody of [1] with a blood cell sample        predicted to comprise a monocyte, and    -   (2) collecting a blood cell that has bound to the antibody in        step (1);-   [4] the method of [2] or [3], wherein the blood cell sample is    peripheral blood, cord blood, or bone marrow;-   [5] a kit for detecting and/or isolating a monocyte, which comprises    the antibody of [1];-   [6] a method for inducing a dendritic cell from a monocyte in vitro,    which comprises the steps of:    -   (1) culturing a monocyte in the presence of a differentiation        inducing factor for a dendritic cell, and    -   (2) contacting a cell cultured in step (1) with the antibody of        [1], detecting HIDE1 expression, and judging that the        differentiation of a monocyte into a dendritic cell is induced        when the expression level of HIDE1 is reduced;-   [7] the method of [6], wherein the differentiation inducing factor    for a dendritic cell is a combination of GM-CSF and IL-4;-   [8] a method for inducing a macrophage from a monocyte in vitro,    which comprises the steps of:    -   (1) culturing a monocyte in the presence of a differentiation        inducing factor for a macrophage, and    -   (2) contacting a cell cultured in step (1) with the antibody of        [1], detecting HIDE1 expression, and judging that the        differentiation of a monocyte into a macrophage-like cell is        induced when the expression level of HIDE1 is reduced;-   [9] the method of [8], wherein the differentiation inducing factor    for a macrophage is phorbol ester;-   [10] a method for obtaining a dendritic cell, which comprises the    steps of:    -   (1) contacting a sample of collected blood cells with the        antibody of [1],    -   (2) collecting a blood cell that has bound to the antibody in        step (1),    -   (3) culturing the blood cell collected in step (2) in the        presence of a differentiation inducing factor for a dendritic        cell,    -   (4) contacting the cell cultured in step (3) with the antibody        of [1], detecting HIDE1 expression, and judging that a monocyte        is differentiated into a dendritic cell when the expression        level of HIDE1 is reduced, and    -   (5) isolating as a dendritic cell the cell judged to be        differentiated in step (4);-   [11] the method of [10], which further comprises the step of    allowing the isolated dendritic cell to ingest an antigen;-   [12] the method of [10], wherein the isolated dendritic cell is used    to prevent and/or treat a tumor.-   [13] the method of [12], which further comprises the step of    allowing the isolated dendritic cell to ingest a tumor-specific    antigen;-   [14] the method of [10], wherein the isolated dendritic cell is used    to prevent and/or treat an autoimmune disease, or to relieve    rejection after an organ transplantation;-   [15] a method for obtaining a macrophage, which comprises the steps    of:    -   (1) contacting a sample of collected blood cells with the        antibody of [1],    -   (2) collecting a blood cell that has bound to the antibody in        step(1),    -   (3) culturing the blood cell collected in step (2) in the        presence of a differentiation inducing factor for a macrophage,    -   (4) contacting the cell cultured in step (3) with the antibody        of [1], detecting HIDE1 expression, and judging that a monocyte        is differentiated into a macrophage-like cell when the        expression level of HIDE1 is reduced, and    -   (5) isolating as a macrophage the cell judged to be        differentiated in step (4);-   [16] the method of [15], which further comprises the step of    activating the isolated cell;-   [17] the method of [15] or [16], wherein the isolated macrophage is    used to treat a spinal cord damage, and/or to treat and/or prevent a    tumor, infectious disease, autoimmune disease, or immunodeficiency    disease;-   [18] a method for collecting a lymphocyte, which comprises the steps    of:    -   (1) contacting the antibody of [1] with a blood cell sample        predicted to comprise a lymphocyte, and    -   (2) collecting a blood cell that did not bind to the antibody in        step (1);-   [19] a method for obtaining an activated lymphocyte, which comprises    the steps of:    -   (1) contacting the antibody of [1] with a blood cell sample        predicted to comprise a lymphocyte,    -   (2) collecting as a lymphocyte a blood cell that is not bound to        the antibody,    -   (3) culturing the lymphocyte collected in step (2), and    -   (4) activating the lymphocyte cultured in step (3) and        collecting the activated lymphocyte;-   [20] the method of [18] or [19], wherein the blood cell sample is    peripheral blood, cord blood, or bone marrow; and-   [21] the method of [19], wherein the activated lymphocyte is used to    prevent and/or treat a tumor or infectious disease.

Monocytes, dendritic cells, macrophages, and lymphocytes can be preparedby using the antibodies of the present invention. Thus, the presentinvention also provides therapeutic and/or preventive methods usingthese cells. Specifically, the present invention relates to uses in theproduction of pharmaceutical compositions to treat and/or prevent tumorsby contacting an antibody of the present invention with a patient bloodcell sample such as peripheral blood, cord blood, or bone marrow;isolating monocytes bound to the antibody; and administering patientswith the dendritic cells obtainable upon treating the monocytes withappropriate cytokines, after activation as required.

Alternatively, the present invention relates to uses of antibodies orfragments comprising a variable region thereof, which bind to proteinsor polypeptides selected from (1) to (3), as antibodies for detectingmonocyte markers. In addition, the present invention relates to uses ofantibodies or fragments comprising a variable region thereof, which bindto proteins or polypeptides selected from (1) to (3), in the productionof kits for detecting and/or isolating monocytes.

-   -   (1) an HIDE1 protein;    -   (2) a protein encoded by a nucleotide sequence that hybridizes        under stringent conditions to a complementary sequence of an        HIDE1 gene; and    -   (3) a polypeptide fragment having at least eight amino acid        residues, which is a fragment of a protein of the above (1) or        (2).

In addition, the present invention provides methods for preventingand/or treating tumors, which comprise the step of administering thosedendritic cells isolated by using an antibody described above.Alternatively, the present invention relates to uses of the dendriticcells isolated using an antibody described above in the production ofpharmaceutical compositions for preventing and/or treating tumors.

Furthermore, the present invention provides methods for preventingand/or treating autoimmune diseases, or relieving rejection after organtransplantation, which comprise the step of administering the dendriticcells isolated using an antibody described above. Alternatively, thepresent invention relates to uses of the dendritic cells, isolated usingan antibody described above, in the production of pharmaceuticalcompositions for preventing and/or treating autoimmune diseases, or forrelieving rejection after organ transplantation.

Moreover, the present invention provides methods for treatment of spinalcord damage and treatment and/or prevention of tumors, infectiousdiseases, autoimmune diseases, and immunodeficiency diseases, whichcomprise the step of administering the macrophages isolated using anantibody described above. Alternatively, the present invention relatesto uses of the macrophages isolated using an antibody described above inthe production of pharmaceutical compositions for treating spinal corddamage and treating and/or preventing tumors, infectious diseases,autoimmune diseases, and immunodeficiency diseases.

In addition, the present invention provides methods for preventingand/or treating tumors or infectious diseases, which comprise the stepof administering the activated lymphocytes isolated using an antibodydescribed above. Alternatively, the present invention relates to uses ofthe activated lymphocytes isolated using an antibody described above inthe production of pharmaceutical compositions for preventing and/ortreating tumors or infectious diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram comparing the amino acid sequences of mouse HIDE1(mHIDE1; SEQ ID NO: 2) and mouse secretory HIDE1 (sHIDE1; SEQ ID NO: 4).Sequence homology was found to be 86.5%. sHIDE1 lacks the transmembranedomain comprised in mHIDE1.

FIG. 2 is a diagram comparing the mRNA sequences of mouse HIDE1 (mHIDE1;SEQ ID NO: 1) and human HIDE1 (hHIDE1; SEQ ID NO: 5). Sequence homologywas found to be 73.9%.

FIG. 3 is a diagram comparing the amino acid sequences of mouse HIDE1(mHIDE1; SEQ ID NO: 2) and human HIDE1 (hHIDE1; SEQ ID NO: 6). Sequencehomology was found to be 68.4%.

FIG. 4 shows the results of confirming acquisition of anti-HIDE1antibody. (a) shows the results of flow cytometry (FCM) using 293Ttransiently expressing hHIDE1 and wild type 293T. The solid lineindicates the reaction of an anti-HIDE1 antibody (1H12) with 293Ttransiently expressing hHIDE1; the broken line indicates the reactionwith wild type 293T. (b) shows the results of Western blotting of293T(H) expressing hHIDE1 and 293T(C) expressing another Myc-tag fusionprotein using an anti-HIDE1 antibody (3F12). An anti-Myc-tag antibody(MBL) was used as the positive control. Arrows indicate the position ofthe positive control bands. (c) shows the results of immunoprecipitationfor 293T(H) expressing hHIDE1 and 293T(C) expressing another proteinusing an anti-HIDE1 antibody (3H3).

FIG. 5 shows the results of (ungated) flow cytometry (FCM) analysis of aperipheral blood mononuclear cell (PBMC) fraction using an anti-HIDE1antibody (1H12). (a) shows the results of two-dimensional fractionationof PBMCs in terms of cell size (FS) and light scattering (SS). From theleft, cell populations were categorized into lymphocytes, monocytes, andgranulocytes. (b) shows the results of staining PBMCs with an isotypiccontrol. (c) shows the results of two-dimensional fractionation of PBMCswith FS and SS. Area M in plot (d) indicates the anti-HIDE1antibody-positive cells. (d) shows the results of FCM analysis of PBMCsstained with the anti-HIDE1 antibody. HIDE1-positive cells were gated,and are shown as “area M”.

FIG. 6 shows the results of double staining with an anti-HIDE1 antibodyand an antibody against CD14, a monocyte marker. Each plot shows thefollowing analysis results:

-   (a) Each cell population separated by double staining was gated (A,    B, and C).-   (b) The whole PBMC was fractionated with FS and SS (gray). Cell    population A from plot (a) (CD14(++) & anti-HIDE1 antibody (+)) is    shown in black (enclosed by the broken line).-   (c) The whole PBMC was fractionated with FS and SS (gray). Cell    population B from plot (a) (CD14(+) & anti-HIDE1 antibody (+)) is    shown in black (enclosed by the broken line).-   (d) The whole PBMC was fractionated with FS and SS (gray). Cell    population C from plot (a) (CD14(−) & anti-HIDE1 antibody (+)) is    shown in black (enclosed by the broken line).

FIG. 7 shows the results of double staining with an anti-HIDE1 antibodyand an antibody against CD11b, a monocyte marker. Each plot shows thefollowing analysis results:

-   (a) Each cell population separated by double staining was gated (A,    B, and C).-   (b) The whole PBMC was fractionated with FS and SS (gray). Cell    population A from plot (a) (CD11b(++) & anti-HIDE1 antibody (+)) is    shown in black (enclosed by the broken line).-   (c) The whole PBMC was fractionated with FS and SS (gray). Cell    population B from plot (a) (CD11b(+) & anti-HIDE1 antibody (+)) is    shown in black (enclosed by the broken line).-   (d) The whole PBMC was fractionated with FS and SS (gray). Cell    population C from plot (a) (CD11b(+) & anti-HIDE1 antibody (−)) is    shown in black (enclosed by the broken line).

FIG. 8 shows the results of double staining with an anti-HIDE1 antibodyand an antibody against CD33, a monocyte marker. Each plot shows thefollowing analysis results:

-   (a) The double positive cell population was gated (gate A).-   (b) The whole PBMC was fractionated with FS and SS (gray). Cell    population A from plot (a) (CD33(+) & anti-HIDE1 antibody (+)) is    shown in black (enclosed by the broken line).

FIG. 9 shows the results of staining cells from human monocytic culturedlines using an anti-HIDE antibody (1H12). (a) used U937, (b) used HL60,and (c) and (d) used THP-1 cultured cells. In (a) to (c), the solid lineshows the reaction using anti-HIDE1 antibody and the broken line showsthe reaction using the isotypic control. In (d), the broken lineindicates the reaction between the anti-HIDE1 antibody and the cellsbefore differentiation, and the solid line indicates the reactionbetween the antibody and the differentiated cells.

FIG. 10 shows the results of double staining human peripheral bloodmononuclear cells (PBMCs) with an anti-hHIDE1 antibody (1H12) and amarker for bone marrow-derived dendritic cell line (BDCA3):

-   (a) double staining of PBMCs with the anti-HIDE1 antibody (1H12) and    an isotype control-   (b) double staining of PBMCs with the anti-HIDE1 antibody (1H12) and    BDCA3.

FIG. 11 shows the results of double staining mouse peripheral bloodmononuclear cells (PBMCs) and mouse spleen cells with an anti-mHIDE1antibody (5F8) and a monocyte marker (CD11b):

-   (a) double staining of mouse peripheral blood mononuclear cells    (PBMCs) with an anti-mHIDE1 antibody (5F8) and an isotype control-   (b) double staining of mouse peripheral blood mononuclear cells    (PBMCs) with an anti-mHIDE1 antibody (5F8) and a monocyte marker    (CD11b)-   (c) double staining of mouse spleen cells with an anti-mHIDE1    antibody (5F8) and an isotype control-   (d) double staining of mouse spleen cells with an anti-mHIDE1    antibody (5F8) and a monocyte marker (CD11b).

FIG. 12 shows the results of double staining mouse peripheral bloodmononuclear cells (PBMCs) and mouse spleen cells using an anti-mHIDE1antibody (5F8) and a dendritic cell marker (CD11c):

-   (a) double staining of mouse PBMCs with an anti-mHIDE1 antibody    (5F8) and an isotype control-   (b) double staining of mouse PBMCs with an anti-mHIDE1 antibody    (5F8) and a dendritic cell marker (CD 11c)-   (c) double staining of mouse spleen cells with an anti-mHIDE1    antibody (5F8) and an isotype control-   (d) double staining of mouse spleen cells with an anti-mHIDE1    antibody (5F8) and a dendritic cell marker (CD11c).

BEST MODE FOR CARRYING OUT THE INVENTION [Antibodies for DetectingMonocyte Markers]

The present invention revealed that monocytes could be more broadlystained using HIDE1 as a marker, than CD14, a known monocyte marker, andalso revealed that more specific staining of monocytes could be achievedwith HIDE1 than with CD11b. Thus, the present invention providesantibodies for detecting monocyte markers (HIDE1). Antibodies that candetect monocytes can be used as the antibodies of the present inventionfor detecting monocyte markers, and such antibodies bind to (1) HIDE1protein, (2) a protein encoded by a nucleotide sequence that hybridizesunder stringent conditions to the complementary sequence of HIDE1 gene,or (3) a polypeptide fragment with at least eight consecutive amino acidresidues of the protein of (1) or (2). Of these, antibodies thatrecognize antigenic regions of HIDE1 protein that are exposed onmonocyte surfaces are particularly preferred as antibodies of thepresent invention for detecting monocyte markers.

Herein, “HIDE1” or “HIDE1 protein” refers to polypeptides encoded by aHIDE1 gene, including isolated natural proteins and recombinant proteinsobtainable by expressing the gene using an appropriate expressionsystem. The nucleotide sequence of mouse HIDE1 gene is shown in SEQ IDNO: 1, and the amino acid sequence of the protein encoded by the gene isshown in SEQ ID NO: 2. Mouse HIDE1 splice variants lacking atransmembrane domain were also found. An amino acid sequence of thesplice variants is shown in SEQ ID NO: 4. The nucleotide sequence of anmRNA encoding the variants is shown in SEQ ID NO: 3. Both thetransmembrane and secretory proteins described above are comprised inthe definition of a HIDE1 protein described herein. The “HIDE1 protein”described herein encompasses not only the two types of mouse protein butalso other variants, isoforms, and homologs of other mammals includinghumans.

The amino acid sequence of human HIDE1 is shown in SEQ ID NO: 6, and thenucleotide sequence encoding human HIDE1 is shown in SEQ ID NO: 5.Isoforms and variants of mouse and human HIDE1, and homologs of othermammals can be prepared, for example, by obtaining genes that encode anisoform, variant, or homolog from an appropriate cDNA library or genomiclibrary using conventional gene cloning techniques such as hybridizationand PCR, using as a probe or primer or the like a gene or a genefragment encoding mouse or human HIDE1; and then expressing the obtainedgenes by known methods.

Nucleotide sequences that hybridize under stringent conditions to acomplementary sequence of a HIDE1 gene have high homology to the HIDE1gene, and are thus expected to encode proteins functionally equivalentto HIDE1. The antibodies of the present invention for detecting monocytemarkers thus comprise antibodies that bind to proteins encoded by suchnucleotide sequences. Furthermore, the antibodies of the presentinvention also comprise antibodies that recognize and specifically bindto an above-described HIDE1 protein or a fragment which is a portion ofa protein encoded by a nucleotide sequence that hybridizes understringent conditions to a complementary sequence of a HIDE1 gene.

Specifically, stringent hybridization conditions of the presentinvention include, for example, conditions whereby hybridization iscarried out using 5×SSC at 25° C. in the absence of formamide.Preferably, hybridization is carried out at 25° C. using 6×SSC in thepresence of 40% formamide. More preferably, hybridization is carried outat 40° C. using 5×SSC in the presence of 50% formamide.Post-hybridization washing is carried out, for example, at 37° C. using2×SSC. Preferably, washing is carried out at 55° C. using 1×SSC. Morepreferably, washing is carried out at 60° C. using 1×SSC.

When an antibody recognizes and binds to a protein, but binding to otherproteins is not substantially detectable under the same conditions, thatantibody can specifically recognize that protein. The degree of bindingbetween an antibody and a protein can be assessed quantitatively basedon immunoassay principles. Specifically, the specificity of an antibodycan be assessed using methods such as FACS and ELISA. When FACS is used,the degree of antibody binding can be compared based on the number ofpositive cells. Alternatively, when ELISA is used, the degree ofantibody binding can be compared based on the signal intensity producedby the labeled antibody. It is safe to conclude that an antibodyspecifically recognizes a target protein when the results of aquantitative evaluation of binding activity show that antibodycross-reactivity between the two proteins is 50% or less, for example,20% or less, typically 10% or less, or 5% or less. For example, when anantibody binds to transformed cells expressing the human HIDE1 gene ofSEQ ID NO: 5, but binding to the host cells used for the transformationis undetectable under the same conditions, the antibody can specificallyrecognize human HIDE1.

Particularly preferable fragments include, for example, fragmentscomprising a hydrophilic region or surface-constituting region of aHIDE1 protein; however, any fragment can be used, as long as it retainsthe antigenicity of a HIDE1 protein or a protein encoded by a nucleotidesequence that hybridizes under stringent conditions to the complementarysequence of a HIDE1 gene. Thus, the antibodies of the present inventionfor detecting monocyte markers comprise antibodies that bind to apolypeptide fragment with eight or more amino acid residues (forexample, 8, 9, 10, 11, 12, or 15 amino acid residues) which is afragment of a HIDE1 protein or a protein encoded by a nucleotidesequence that hybridizes under stringent conditions to the complementarysequence of a HIDE1 gene.

Any antibody can be used as an antibody of the present invention, aslong as it can detect an HIDE1. The antibodies include polyclonalantibodies, monoclonal antibodies, chimeric antibodies such as humanizedantibodies, antibody fragments comprising a variable region (Fab, Fab′,F(ab′)2, Fv, and such), multispecific antibodies, and single-chainantibodies (scFv). The antibodies of the present invention may bemodified by PEG or the like, as required. Further, as required, it ispossible to label the antibodies with an appropriate enzyme(acetylcholine esterase, alkaline phosphatase, β-galactosidase,glucose-6-phosphate dehydrogenase, peroxidase, maltose-binding enzyme,glutathione transferase, and such), biotin, green fluorescence protein(GFP), radiolabel, fluorescent label, or the like, so that detection canbe achieved without using a secondary antibody. When labeled withbiotin, the antibodies can be recovered based on the binding betweenbiotin and avidin, streptavidin, or the like. When the antibodies arefluorescently labeled, cells bound to the antibodies can be separated bya cell sorter using the fluorescence signal as an indicator. When boundto magnetic particles, the antibodies can be separated using a magneticfield or magnet. Further, if required, the antibodies of the presentinvention may be bound to an appropriate solid phase carrier, or thelike.

The antibodies of the present invention can be produced by knownmethods. For example, when appropriate immune animals are immunized withHIDE1 protein or an antigenic fragment thereof, polyclonal antibodiesrecognizing HIDE1 can be obtained from the immune animals. The immuneanimals used for the purpose described above are not particularlylimited, and any animal may be used as long as it can produce anantibody of the present invention. In general, animals belonging toRodentia (mice, rats, hamsters, and such), Lagomorpha, Primates(cynomolgus monkeys, Rhesus monkeys, hamadryas baboons, chimpanzees, andsuch) are used to produce antibodies. Alternatively, for obtaining humanantibodies, transgenic animals having the repertoire of human antibodygenes may be used as immune animals.

Antigens used to prepare the antibodies of the present invention includethe above-described (1) HIDE1 proteins, (2) proteins encoded by anucleotide sequence that hybridizes under stringent conditions to acomplementary sequence of a HIDE1 gene, and (3) polypeptide fragmentswith at least eight consecutive amino acid residues from a protein ofthe above (1) or (2). Particularly preferable antigen fragments are, forexample, portions of HIDE1 proteins that are exposed on monocytesurfaces. For example, in the amino acid sequence of human HIDE1 shownin SEQ ID NO: 6, the extracellular domain corresponds to the amino acidsequence from position 27 to 117. Thus, polypeptides comprising theamino acid sequence from position 27 to 117 of human HIDE1 sequence, andpolypeptides comprising an amino acid sequence of eight consecutiveamino acids or more selected from the amino acid sequence from position27 to 117 of human HIDE1 sequence are preferred antigen fragments.

Such antigens can be administered to immune animals, for example, byinjection or such, in combination with an adjuvant if required, toimmunize the animals. For immunization, antigens are preferablyadministered several times at fixed intervals. Sera are collected fromthe immunized animals and may be used as polyclonal antibodies orfurther purified if required.

It is also possible to prepare monoclonal antibodies by cloningantibody-producing cells from animals confirmed to produce an antibodywith a desired activity. More specifically, spleens are excised fromimmunized animals; immune cells are isolated from the spleens; the cellsare immortalized; and cells producing a desired monoclonal antibody areselected from the cells. A standard method for immortalizingantibody-producing immune cells comprises preparing hybridomas by fusingthe cells with appropriate myeloma cells (see, for example, MethodsEnzymol. (1981) 73: 3-46). Methods that immortalize cells by introducingan oncogene are also known. The obtained antibody-producing cells can becultured and the culture supernatant yielded can be used as a monoclonalantibody or purified as required. Alternatively, the cells confirmed toproduce a desired monoclonal antibody can be intraperitoneallytransplanted to mice or such to collect desired monoclonal antibodiesfrom the mice ascites by.

Alternatively, antibodies can be prepared by cloning antibody-encodinggenes from the obtained antibody-producing cells using geneticengineering techniques. Once the antibody-encoding genes are cloned,chimeric antibodies, humanized antibodies, multispecific antibodies,scFv, and the like can be prepared. Such recombinant antibodies are alsocomprised in the antibodies of the present invention. Further, theantibodies of the present invention for detecting monocyte markers alsocomprise antibody fragments. Such antibody fragments can be produced bytreating the polyclonal or monoclonal antibodies described above with anenzyme such as papain or pepsin. Alternatively, such antibody fragmentscan be obtained by preparing a polynucleotide chain that encodes anantibody fragment and inserting it into an expression vector, thenexpressing the fragment using conventional methods.

The antibodies of the present invention, comprising antibody fragments,can be collected and purified using protein A, protein G, or such. Theantibodies can also be purified by using combinations of standardprotein purification methods (ethanol precipitation, salting out,various types of chromatographic methods, gel electrophoresis, gelfiltration, ultrafiltration, recrystallization, acid extraction,distillation, dialysis, immunoprecipitation, solvent extraction, solventprecipitation, ammonium sulfate precipitation, and such). Theconcentration of a yielded antibody can be determined by known methodssuch as absorbance measurements and enzyme-linked immunosorbent assays(ELISAs). Further, the antigen-binding activity of an antibody can bedetermined by absorbance measurements, fluorescence antibody methods,enzyme immuno assays (EIAs), radioimmunoassays (RIAs), ELISAs, or such.The activity of an antibody can also be evaluated using commerciallyavailable assay systems such as BIAcore (Pharmacia).

[Detection and/or Isolation of Monocytes]

Since the antibodies of the present invention bind to monocytes, theycan be used to detect monocytes in a sample or to obtain (isolate)monocytes. Specifically, the present invention provides methods fordetecting and isolating monocytes. Monocytes can be detected bycontacting an antibody of the present invention with a blood cell samplepredicted to contain monocytes, and then detecting cells that bind tothe antibody. Meanwhile, monocytes can be isolated by contacting anantibody of the present invention with a blood cell sample predicted tocontain monocytes, and then collecting cells that bind to the antibody.

The methods of the present invention for detecting monocytes can be usedin the diagnosis of diseases in which monocytes are involved. Forexample, since monocytes are known to increase in the circulatory systemin inflammatory diseases, post-transplantation rejection, monocyticleukemia, infectious disease recovery phases, or such, such symptoms canbe diagnosed by detecting monocytes in the peripheral blood or such.Further, since monocytes can differentiate into macrophages, dendriticcells, and the like, monocytes isolated by the methods of the presentinvention can be used in cell immunotherapy or such after beingdifferentiated into macrophages and dendritic cells.

Herein, a “blood cell sample” can include, but is not limited to, forexample, bone marrow, peripheral blood, and cord blood. A particularlypreferred blood cell sample is peripheral blood. Such blood cell samplescan be obtained from appropriate individuals. Herein, an individualrefers to an individual whose tissue differentiation is completed andthat can survive independently of its mother, and includes individualsat every growth stage, including not only adults but also individualsimmediately after birth. It is not important whether the individualsfrom which the blood cell samples used in the present invention arecollected are dead or alive, as long as proliferative cells can beseparated from them. Thus, blood cell samples used in the presentinvention can be obtained from living individuals or individuals in astate of brain death or cardiac arrest.

Individuals for obtaining blood cell samples are vertebrates, forexample, humans, mice, rats, rabbits, chickens and so on. Humans andmice are particularly preferred subjects. When aiming to use isolatedmonocytes in cell immunotherapy, for example, the blood cell sample usedto isolate the monocytes is preferably collected from the actual patientto be treated. The risks of rejection reactions and infection byinfectious pathogens can be reduced by using the patient's own cells.

The binding reaction between HIDE1 and the antibodies can be detectedusing immunoassay principles. In typical immunoassays, either protein orantibody is immobilized and detection is achieved after separatingunreacted components. Methods for immobilizing cells or antibodies areknown. For example, they can be immobilized directly onto solid phase bychemical linking or physical adsorption. Alternatively, when an antibodyof the present invention is biotinylated, it is also possible toindirectly immobilize the antibody onto a solid phase which has adsorbedavidin, streptavidin, or the like. When an antibody is bound to magneticparticles, not only the antibody but also cells bound to the antibodycan be quickly and conveniently detected and isolated using a magnet.Alternatively, when using an antibody that recognizes multiple antigens,such as a multispecific antibody, the antibody is bound to the HIDE1 onmonocytes, and then other antigens can be bound to the antibody.Alternatively, antibodies can be immobilized onto a solid phase viaprotein A or G or the like.

The timing of antibody immobilization is not particularly limited and anantibody may be immobilized before, after, or simultaneously uponcontact with a sample. An arbitrary solid phase may be used toimmobilize the antibodies. Such solid phases include membranous,particulate, or fibrous carriers, which are made of glass; organicpolymers such as polystyrene; and inorganic materials such as silicagel, alumina, and activated carbon. For example, the antibodies of thepresent invention may be immobilized onto the inner wall of a reactioncontainer such as a plate, dish, and test tube, or to a bead.

Immunological methods for detecting or quantifying monocytes using theantibodies of the present invention for detecting monocyte markersinclude, for example, fluorescence antibody methods (see MonoclonalAntibodies: Principle and Practice, 3rd ed. (1996) Academic Press),ELISAs, RIAs, immunohistochemical staining such as immunocytologicalstaining and immunohistological staining, (see, for example, the ABCmethod and CSA method; Monoclonal Antibodies: Principle and Practice,3rd ed. (1996) Academic Press), Western blotting, andimmunoprecipitation.

In ELISA, an antibody is labeled with an enzyme such as a peroxidase,whose substrate can be detected easily, or which catalyzes a reactiongenerating a detectable product, and then the concentration of thesubstrate or product or such is determined using an absorptiometer afterreacting the enzyme with the substrate. Sandwich ELISA, which is oneELISA method, uses two types of antibodies that bind to differentantigen epitopes, where one of the two is labeled with an enzyme. InRIAs, an antibody is radiolabeled so that it can be detected andquantified using a scintillation counter. In immunohistochemicalstaining, tissues, cells, or the like are reacted with an antibodylabeled with a fluorescent substance or enzyme or such, and then pigmentor such produced by the fluorescent or enzymatic reaction is observedusing a microscope to investigate the tissue or cellular localization ofa substance with which the antibody reacts. In immunoprecipitation, anantibody is reacted to cells, and then a carrier that specifically bindswith immunoglobulin, such as protein G-Sepharose, is added to thereaction solution to precipitate the antigen-antibody complex.

A particularly preferred method for detecting and/or isolating monocytescomprises detecting and isolating cells of interest with a cell sorterusing an antibody fluorescently labeled with fluorescein isothiocyanate(FITC), phycoerythrin, or such, using the fluorescence signal as anindicator. When antibodies that bind to different cell surface antigensare labeled with dyes with different fluorescent wavelengths and used incombination, cells can be selected using multiple cell surface antigens.In an alternative preferred method, cells of interest can be capturedonto magnetic particles by reacting cells with antibody-immobilizedmagnetic particles. The cells bound to magnetic particles are separatedusing a magnetic device such as MACS (Daiichi Pure Chemicals Co.), andthen the cells of interest can be collected. Such methods, comprisingselecting cells using a single cell surface antigen and separating themusing magnetic particles, are simple, and therefore particularlypreferred as the methods of the present invention for detecting and/orisolating monocytes.

Isolated cells can be preferably cultured in any of various culturemedia (RPMI, IMDM, and such) known to those skilled in the art,supplemented with serum and amino acids. The culture is preferablyconducted at 37° C. under sterile conditions. Other culture condition scan be determined appropriately by those skilled in the art, dependingon the purposes and such of the cells.

CD14 is known as a monocyte marker, and dendritic cells derived fromCD14-positive monocytes are also being used in cell immunotherapy (Picklet al., J. Immunol. (1996) 157:3850-9; Jefford et al., Blood (2003) 102:1753). When monocytes are differentiated into dendritic cells,expression levels of both cell-surface CD14 and HIDE1 are reduced,although HIDE1 expression clearly fades more rapidly (two to three daysafter induction of differentiation) than CD14 expression. Specifically,there is a CD14-negative, HIDE1-positive stage in the process ofdifferentiation from monocytes to dendritic cells. Thus it is suggestedthat the HIDE1 of the present invention may be expressed morespecifically in monocytes than the conventional monocyte marker, CD14.Therefore, the methods of the present invention for detecting and/orisolating monocytes using HIDE1 as an indicator are expected to yield apurer monocyte population. In one embodiment, the present inventionprovides a monocyte population isolated using HIDE1 as a marker, wherethe monocyte population is purer than that yielded using a conventionalmarker.

[Kits]

The antibodies of the present invention for detecting monocyte markerscan form monocyte detection or isolation kits, in combination withsubstances required for detecting and/or isolating monocytes, and so on.For example, the antibodies of the present invention can form such kits,in combination with reagents, devices, and the like required to detectantibodies. For example, the kits may include: secondary antibodies fordetecting the antibodies of the present invention; when the antibodiesof the present invention are labeled with an enzyme,the kits may includethe substrate in the reaction catalyzed by the enzyme; and when theenzyme has been bound to magnetic particles, the kits may include amagnet or such. Furthermore, in combination with the antibodies of thepresent invention, media suitable for monocyte culture, cytokinesrequired for differentiation from monocytes to macrophages or dendriticcells, and others may constitute the kits. A manual is preferablyattached to the kits, describing directions for use of the antibodiesincluded in the kits for detecting and isolating monocytes, for theculture of the isolated monocytes, and for the induction ofdifferentiation, and so on.

[Induction of Monocyte Differentiation]

The action of appropriate cytokines is known to differentiate monocytesinto macrophages (Nature (1987) 325:262-5; Nature (1986) 323: 86-9; J.Immunol. (1988) 140: 1345-9; Jpn. J. Cancer Res. (1989) 80: 59-64),dendritic cells (see, for example, Thurnher et al., Exp. Hematology(1997) 25: 232-7; Schuler and Steinman, J. Exp. Med. (1997) 186:1183-7), osteoclasts (Lacey et al., Endocrinol. (1988) 136: 2367-76;Mundy, Bone and Miner. Res. (1993) 8: S505-10; Japanese PatentApplication Kokai Publication (JP-A (Kokai)) H11-196864 (unexamined,published Japanese patent application), or such. Differentiation of themonocytes isolated using the antibodies of the present invention fordetecting monocyte markers can also be induced by the known techniques.Thus, in one embodiment, the present invention provides methods thatcomprise isolating monocytes using an antibody of the present invention,and then differentiating the isolated monocytes into macrophages,dendritic cells, osteoclasts, and such.

The present invention also revealed that HIDE1 expression was reduced orabolished in dendritic cells derived and differentiated from monocytesof human peripheral blood using GM-CSF and IL-4, and macrophage-likecells differentiated from THP-1, a monocytic cultured cell line, byphorbol ester, a differentiation-inducing factor. Thus, thedifferentiation of monocytes into macrophages or dendritic cells can beconfirmed by monitoring the expression level of HIDE1 during in vitroinduction of macrophages or dendritic cells from monocytes. Therefore,the present invention provides methods for inducing macrophages ordendritic cells from monocytes, which comprise confirming celldifferentiation using HIDE1 expression level.

The methods of the present invention for inducing dendritic cells frommonocytes in vitro comprise the steps of: (1) culturing monocytes in thepresence of a differentiation inducing factor for dendritic cells, and(2) detecting HIDE1 expression in the cultured cells using an antibodyof the present invention. In addition, the methods of the presentinvention for inducing macrophages from monocytes in vitro comprise thesteps of: (1) culturing monocytes in the presence of a differentiationinducing factor for macrophages, and (2) detecting HIDE1 expression inthe cultured cells using an antibody of the present invention. In bothmethods, a reduced HIDE1 expression level in step (2) indicates themonocytes are differentiated into dendritic cells or macrophages.

Dendritic cells (DCs) can be induced from monocytes by known methods(see, for example, J. LeukocyteBiol. (1996) 59: 208-18; Thurnher et al.,Exp. Hematology (1997) 25: 232-7; Schuler and Steinman, J. Exp. Med.(1997) 186: 1183-7). Cytokines used to induce the differentiation ofmonocytes into DCs by the known methods can also be used asdifferentiation inducing factors for dendritic cells in the methods ofthe present invention for inducing DCs from monocytes. In particular, ithas been reported that human monocytes cultured in the presence ofgranulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4 havethe ability to ingest antigens (phagocytosis) and transmit that antigeninformation to T cells, thereby activating the T cells (J. LeukocyteBiol. (1996) 59: 208-18). Thus, the combination of GM-CSF and IL-4 isparticularly preferred as a differentiation inducing factor for adendritic cell in the methods of the present invention.

DCs are classified into immature and mature DCs, according to theirdifferentiation stages. Mature dendritic cells are characterized by theinduction of T cell growth as well as the expression of CD80, CD86,CD83, MHC-I, MHC-II and so on. The phagocytic activity is stronger inimmature DCs and weaker in mature DCs. The ability to present antigensto T cells accords with the expression levels of CD40, CD80, CD86,MHC-I, and MHC-II, which are involved in this ability. The ability isweak in immature DCs, but stronger in mature DCs. DCs confirmed to havedifferentiated from monocytes using an antibody of the present inventioncan also be further classified using these properties and/or markers bywhich immature and mature DCs are discriminated as indicators.

It has been reported that cytotoxic macrophages can be induced byculturing monocytes isolated from human peripheral blood in a culturemedium containing serum in the presence of IL-1, IL-2, tumor necrosisfactor (TNF), interferon γ(IFN-γ), or the like for 18 to 48 hours(Nature (1987) 325: 262-5; Nature (1986) 323: 86-9; J. Immunol. (1988)140: 1245-9; Jpn. J. Cancer Res. (1989) 80: 59-64). JP-A (Kokai)H05-130863 reported that extremely cytotoxic macrophages could beyielded by using IL-2 and IL-1 or CSF-1 in combination. Cytokines usedto induce the differentiation of monocytes into macrophages in theseknown methods can also be used as differentiation inducing factors formacrophages in the methods of the present invention for inducingmacrophages from monocytes. Phorbol ester, used in Examples, is aparticularly preferred differentiation inducing factor for macrophagesin the methods of the present invention.

The monocytes used to induce dendritic cells and macrophages are notparticularly limited, and include monocytic cultured cell lines THP-1,HL60, U937, and the like, as well as monocytes obtained from peripheralblood, cord blood, bone marrow, and such. The differentiation ofmonocytes is preferably induced in various culture media (RPMI, IMDM,and such) known to those skilled in the art, which are supplemented withserum and amino acids. The cells are preferably cultured under sterileconditions at 37° C. Induction of the differentiation of monocytes intodendritic cells is known to take five to seven days, and induction ofthe differentiation into macrophages is known to take 18 to 24 hours.Therefore, it is preferable to continue the culture for a period of fivedays or longer to achieve the differentiation into dendritic cells, orfor 18 hours or longer to achieve the differentiation into macrophages.Other culture conditions can be determined appropriately by thoseskilled in the art depending on the purpose and such of the cells.

[Methods for Obtaining Dendritic Cells and Macrophages]

Dendritic cells and macrophages can be obtained from blood cell samplesby using the above-described methods for isolating monocytes and forinducing dendritic cells or macrophages from monocytes. Specifically,the present invention provides methods for obtaining dendritic cells andmacrophages. It is known that dendritic cells can be used to preventand/or treat tumors, autoimmune diseases such as rheumatism, or torelieve rejection reactions after organ transplantation, and dendriticcells obtained by the methods of the present invention can also be usedfor these purposes.

The use of macrophages has been proposed to treat spinal cord damage, orto treat and prevent tumors, infectious diseases, autoimmune diseases,and immunodeficiency diseases. Macrophages obtained by the methods ofthe present invention can also be used for these kinds of purposes.Further, it is known that macrophages have strong biosynthetic abilitiesand secrete biologically active, important macromolecules, includingcytokines, growth factors, inflammatory mediators, proteases, andprotease inhibitors. In addition, macrophages at a given stage ofdifferentiation have only a limited lifetime and also have the propertyof migrating to inflammation sites and such, and thus it has also beenproposed that such macrophages can be candidate cells for gene therapy(Japanese Patent Kohyo Publication No. (JP-A (Kohyo)) 2001-504683(unexamined Japanese national phase publication corresponding to anon-Japanese international publication). Macrophages obtained by themethods of the present invention can also be used as such cells for genetherapy.

The methods of the present invention for obtaining dendritic cells (DCs)comprise the steps of: (1) culturing blood cells in the presence of adifferentiation inducing factor for DCs; (2) contacting the culturedcells with an antibody for HIDE1 marker detection of the presentinvention, detecting HIDE1 expression, and judging that monocytes aredifferentiated into DCs when the HIDE1 expression level is reduced; and(3) isolating cells judged to be differentiated into DCs. The onlyrequirement is that the blood cells to be differentiated into DCscomprise monocytes. However, to obtain more homogeneous DC populations,it is preferable to use isolated monocytes as a starting material.

Preferred blood cells comprising monocytes can be prepared, for example,by (1) contacting a blood cell sample with an antibody of the presentinvention for detecting a HIDE1 marker, and then (2) collecting bloodcells bound to the antibody. Blood cell samples to be used for thispurpose are not particularly limited, and any blood cell sample may beused as long as it comprises monocytes. Such blood cell samples include,for example, bone marrow, peripheral blood, and cord blood; andparticularly preferably include peripheral blood. Such blood cellsamples can be collected from appropriate individuals. However, when DCsprepared from isolated monocytes are administered to patients fortreating or preventing disease, it is preferable to use a blood cellsample collected from the patient being administered, to avoid the riskof rejection and infection by infectious pathogens.

Thus, in one embodiment, the present invention provides methods forobtaining DCs, which comprise the steps of: (1) contacting a sample ofcollected blood cells with an antibody of the present invention; (2)collecting blood cells bound to the antibody; (3) culturing thecollected blood cells in the presence of a differentiation inducingfactor for DCs; (4) contacting the cultured cells with an antibody ofthe present invention, detecting HIDE1 expression, and judging thatmonocytes are differentiated into DCs when the expression level of HIDE1is reduced; and (5) isolating the cells judged to be differentiated intoDCs.

DCs are classified into immature and mature DCs according to theirdifferentiation stages. Mature DCs are characterized by induction of Tcell growth, and the expression of CD80, CD86, CD83, MHC-I, MHC-II andso on. The phagocytic activity is stronger in immature DCs but weaker inmature DCs. The ability to present antigens to T cells accords with theexpression levels of CD40, CD80, CD86, MHC-I, and MHC-II, which areinvolved in this ability. The ability is weak in immature DCs, butstronger in mature DCs. DCs confirmed to have differentiated frommonocytes using an antibody of the present invention can also be furtherclassified using these properties and/or markers by which immature andmature DCs are discriminated as indicators.

In immune responses that occur upon invasion of a foreign antigen intothe living body, DCs phagocytose the foreign antigens, migrate intolymph nodes, and present the antigens to lymphocytes. Thus, diseasesinvolving the invasion of foreign antigens can be treated by injectingimmature DCs directly into areas where the foreign antigens exist in thebody. Therapeutic methods comprising injecting DCs directly into lesionssuch as tumors (dendritic cell injection (DCI)), are being performed,and DCs obtained by the methods of the present invention can also beused to treat tumors by direct injection into such lesions.

In addition, methods are known that use immature DCs to treat andprevent autoimmune diseases such as rheumatism, and to relieve rejectionin organ transplantation, and such. For example, it is believed that theadministration of immature DCs can result in IL-10 production ordifferentiation of IL-10-producing suppressor T cells from naive Tcells. IL-10 has the activity of inducing anergy (unresponsiveness) in Tcells that attack autologous cells or organs derived from donors. Morespecifically, IL-10 has the activity of suppressing the development andactivity of type 1 helper T cells and killer cells. It is understoodthat when immature DCs are administered, damage to autologous tissuesand graft rejection is relieved based on this mechanism. DCs obtained bythe methods of the present invention can also be used in the methodsdescribed above.

Further, methods using the DCs′ antigen presenting ability are alsoknown, whereby DCs ingest antigens in vitro prior to theiradministration to the body; and then these DCs are used in cellimmunotherapy. Thus, in one embodiment of the present invention, themethods for obtaining DCs include methods further comprising the step ofallowing cells, which have been judged to be differentiated into DCs, toingest an antigen. DCs can ingest the antigen by contacting the antigenwith the DCs, for example, by adding it to the DC culture medium. Acytokine such as TNF-α can also be added to the medium when contactingDCs with antigen molecules. To ensure antigen presentation by DC,antigens are contacted with DCs for several hours to several days,preferably for four to 48 hours. During this contact the medium may bechanged or cytokines and antigens may be further added as required.

The antigens to be introduced into DCs include antigen componentsobtained from patients and known antigens produced artificially. Forexample, if aiming to suppress cancer recurrence and metastasis, anantigen extracted from an autologous cancer (a cancer cell lysate,acid-extracted peptide, or such) may be used as the antigen to beingested by the DCs; or when there is a known tumor-specific antigenassociated with a target cancer, an artificially produced tumor-specificantigen polypeptide may be used as the antigen to be ingested by theDCs. When aiming for cancer prevention, known tumor-specific antigensmay be used. Alternatively, when aiming for cancer treatment,tumor-specific antigens or antigens extracted from an autologous cancer(cancer cell lysate, acid-extracted peptide, or such) can be used.

The present inventors found that, in addition to monocytes, bonemarrow-derived dendritic cells could also be isolated by collectingHIDE1-positive cells from human peripheral blood samples. Specifically,not only monocytes but also dendritic cells can be collected when HIDE1is used as a marker to obtain these precursor monocytes for inducingdendritic cells. It is a novel finding obtained by the present inventorsthat peripheral blood contains HIDE1-positive bone marrow-deriveddendritic cells, although in small numbers. Specifically, the presentinvention provides methods for isolating and/or detecting monocytes andbone marrow-derived dendritic cells in peripheral blood, which comprisethe steps of:

-   (1) contacting a peripheral blood sample with an anti-HIDE1    antibody; and-   (2) collecting the blood cells that bound to the antibody in step    (1), which comprise monocytes and bone marrow-derived dendritic    cells.

Alternatively, the present invention provides reagents comprisinganti-HIDE1 antibodies, which are used to isolate and/or detect monocytesand bone marrow-derived dendritic cells in peripheral blood. The presentinvention also relates to uses of anti-HIDE1 antibodies to producereagents for isolating and/or detecting monocytes and bonemarrow-derived dendritic cells in peripheral blood.

The methods of the present invention for obtaining macrophages comprisethe steps of: (1) culturing blood cells in the presence of adifferentiation inducing factor for macrophages; (2) contacting thecultured cells with an antibody of the present invention for detecting aHIDE1 marker, detecting HIDE1 expression, and judging that monocytes aredifferentiated into macrophage-like cells when the expression level ofHIDE1 is reduced; and (3) isolating the cells judged to havedifferentiated into macrophage-like cells. The only requirement is thatthe blood cells to be differentiated into macrophages comprisemonocytes. However, to prepare more homogeneous macrophage populations,it is preferable to use isolated monocytes as a starting material.

Preferred blood cells comprising monocytes can be prepared, for example,by (1) contacting a blood cell sample with an antibody of the presentinvention for detecting a HIDE1 marker, and then (2) collecting bloodcells bound to the antibody. Blood cell samples to be used for thispurpose are not particularly limited, and any blood cell sample may beused as long as it comprises monocytes. The blood cell samples include,for example, bone marrow, peripheral blood, and cord blood; andparticularly preferably include peripheral blood. Such blood cellsamples can be collected from appropriate individuals.

However, when macrophages prepared from isolated monocytes areadministered to patients for treating or preventing disease, it ispreferable to use a blood cell sample collected from the patient beingadministered, to avoid the risk of rejection and infection by infectiouspathogens. Thus, in one embodiment, the present invention providesmethods for obtaining macrophages, which comprise the steps of: (1)contacting a sample of collected blood cells with an antibody of thepresent invention; (2) collecting blood cells bound to the antibody; (3)culturing the collected blood cells in the presence of a differentiationinducing factor for macrophages; (4) contacting the cultured cells withan antibody of the present invention, detecting HIDE1 expression, andjudging that monocytes are differentiated into macrophage-like cellswhen the expression level of HIDE1 is reduced; and (5) isolating thecells judged to be differentiated into macrophages.

It is known that macrophages have strong biosynthetic ability, secretebiologically active, important macromolecules, including cytokines,growth factors, inflammatory mediators, proteases, protease inhibitors,active oxygen, and nitrogen monoxide, and have cytotoxicity againsttumor cells, phagocytotic activity, antimicrobial activity, and such.Since macrophages have these kinds of characteristics, adoptiveimmunotherapy has been proposed, in which macrophages are activated exvivo before being administered to patients. In one embodiment of thepresent invention, the methods for obtaining macrophages furthercomprise activating cells judged to be differentiated into macrophages.Macrophage-activation factors that are known to activate the functionsof macrophages can be used to activate macrophages. Macrophages can beactivated, for example, by adding a macrophage activation factor such asIFN-γ, IFN-α/β, IL-1, TNF, GM-CSF, or macrophage migration inhibitoryfactor (MIF), to the medium.

[Collection and Acquisition of Lymphocytes]

The present invention revealed that HIDE1 is mainly expressed inmonocytes but not in lymphocytes. Thus, lymphocytes can be collectedfrom samples such as peripheral blood, which comprise lymphocytes andmonocytes, by removing the monocytes from the sample by binding with theanti-HIDE1 antibodies of the present invention. Specifically, thepresent invention provides methods for collecting lymphocytes, whichcomprise the steps of: (1) contacting an antibody of the presentinvention with a blood cell sample predicted to contain lymphocytes, and(2) collecting blood cells that are not bound to the antibody.

Lymphocytes collected by the methods of the present invention can beused to prevent and treat tumors and infectious diseases, especiallyviral infections and the like, where lymphocytes are known to beeffective. Known methods of adoptive immunotherapy comprise activatingex vivo autologous lymphocytes obtained from patients and thenadministering them to the patients. Thus, in one embodiment of thepresent invention, lymphocytes collected using an antibody of thepresent invention can be activated ex vivo to prepare activatedlymphocytes.

The blood cell samples from which lymphocytes are collected are notparticularly limited, and any blood cell sample can be used as long asit comprises monocytes. Such blood cell samples include, for example,bone marrow, peripheral blood, and cord blood; peripheral blood isparticularly preferable. Such blood cell samples can be collected fromappropriate individuals. However, when collected lymphocytes areadministered to a patient to treat or prevent diseases, it is preferableto use a blood cell sample collected from the patient beingadministered, to avoid the risk of rejection reaction and infection byinfectious pathogens. Various cytokines are known to have the activityof activating lymphocytes. For example, it is known thatlymphokine-activated killer (LAK) cells with strong antitumor activityare yielded by reacting peripheral blood lymphocytes with IL-2(activated lymphocyte therapy; J. Immunol. (1984) 132: 2123-8; J. Exp.Med. (1982) 155: 1823-41). Thus, LAK cells can be obtained by using IL-2to activate lymphocytes collected by the methods of the presentinvention.

[Administration of Cells]

Monocytes, dendritic cells, macrophages, and lymphocytes obtained by theabove-described methods of the present invention can be administered topatients to treat and prevent various diseases, to improve QOL, or forother purposes. Thus, the present invention provides methods foradministering patients with one or more of these cells, therapeutic andpreventive methods using these cells, and uses of these cells relatingto treatment and prevention. Such cells isolated, obtained, or collectedby any of the methods described above are cultured, amplified, and/oractivated, if required. Cells of interest can be collected and, ifrequired, washed, fractionated, concentrated, and so on, and thenadministered to patients. The cells are administered to appropriatesites, selected depending on each case and cell type, and areadministered intravenously, intraperitoneally, intrathoracically,intratumorally, intraarterially, or by other route. Those skilled in theart can adjust the dose of each cell based on the patient's physicalconstitution, sex, age, symptoms and such, and the type of cell. Thecells may be suspended in an appropriate vehicle when administered topatients. Preferred vehicles used to suspend the cells includephysiological salines.

Diseases targeted by activated lymphocyte (LAK) therapies includecancers and malignant tumors such as sarcomas, and the therapies areperformed to prevent recurrence (prevention of recurrence afterextirpation of cancer lesions by surgical treatment), to treat diseases,and to improve QOL. Furthermore, the therapies have also been found tobe effective against viral infections such as hepatitis C and B, and arealso used against viral infections to potentiate patient immunity,prevent cancer development, and so on. Specifically, an appropriateamount of blood (about 15 ml) is collected from patients, lymphocytesare removed according to the methods of the present invention, thelymphocytes are amplified as much as possible by about two weeks ofculture, and activated lymphocytes are returned to the patient's body.

When aiming to prevent recurrence of malignant tumors after extirpationof lesions, LAK cells are administered six times at about two-weekintervals, and then monthly for two to five years. The therapies canalso be applied to recurrent cancers and cancers that are not operablebecause of their locations. In such cases, LAK cells are administeredapproximately weekly, and if effects are observed, administration ispreferably continued. Alternatively, LAK therapy can be used alongsidetreatments using anti-cancer agents and so on to produce synergisticeffects and reduce adverse effects. When the therapies are used incombination with anti-cancer agents and so on, it is preferable tocollect the blood required for the therapy prior to carrying out theother treatment. Lymphocytes collected or activated by the methods ofthe present invention may also be administered to patients using similarprocedures to those conventionally used to administer lymphocytes.

Dendritic cell (DC) therapies are also used to prevent and treatrecurrences of cancers and malignant tumors such as sarcomas. DCs arealso important for conferring tolerance to rejection after organtransplantation and to autoimmune diseases. Methods used to achieve DCimmunotherapy comprise administering cells obtained from a patient'sblood or such directly into lesions of tumors or such, or allowing DCsto ingest tumor-specific antigens. The methods for allowing DCs toingest tumor-specific antigens comprise providing components extractedfrom tumor cells obtained from patients, or providing knownartificially-produced tumor-specific antigens.

In particular, these therapies are used to prevent recurrence afterextirpation of cancers by surgery, or to treat metastatic cancers to theliver, lung, lymph nodes, and under the skin or such, as well asrecurrent cancers. Basically, DCs which have ingested antigens areinjected intradermally into areas surrounding the lymph nodes. Theintrathoracic or intraperitoneal route may be selected depending on thetype, location and such of the tumor. When the cells are administereddirectly into lesions of tumors or the like, CTs may be used asrequired. Some reports describe the use of dendritic cells to treat andprevent gastrointestinal cancers, malignant melanoma, hematopoietictumors such as B cell tumor and chronic myelogenous leukemia, breastcancer, and the like. The cell dose used previously for directintratumoral administration of DCs is about 1×10⁸ cells. DCs prepared bythe methods of the present invention can also be administered topatients using similar procedures to those conventionally used foradministering DCs.

Macrophages are being used to treat and/or prevent tumors, infectiousdiseases (particularly viral infections), autoimmune diseases, andimmunodeficiency diseases. Furthermore, macrophage-based therapies forspinal cord damage are also known. Macrophages obtained by the methodsof the present invention can also be administered to patients usingsimilar procedures to those conventionally used for administeringmacrophages.

Hereinbelow, the present invention will be specifically described usingExamples.

All the prior art documents cited herein are incorporated by reference.

EXAMPLES 1. Preparation of Anti-HIDE1 Antibodies

Monoclonal antibodies against a human homolog of HIDE1 were prepared bythe following procedure: First, PCR was used to amplify a human HIDE1gene from a placental cDNA library. The yielded PCR product was clonedinto pcDNA3.1 (Invitrogen), an expression vector for cultured animalcells (pcDNA-hHIDE1). In the constructed vector, the C terminus of humanHIDE1 (hHIDE1) is fused with a Myc tag. Cells of the human cultured cellline 293T were transiently transfected with pcDNA-hHIDE1 usingLipofectamine 2000 (Invitrogen), and the yielded cells expressing hHIDE1were used as antigens to immunize Balb/C mice. Lymphocytes collectedfrom the lymph nodes of the immunized mice were fused with Myeloma P3U1.After ten days, supernatant from each clone cell was collected assamples. Cell supernatants that reacted with the hHIDE1 -expressing cellline were selected by flow cytometry (FCM). Thus, hybridomas producinganti-HIDE1 antibodies were obtained.

2. Flow Cytometry (FCM)

hHIDE1-expressing 293T and wild type 293T were each suspended in PBScontaining 2 mM EDTA and 0.5% BSA, and were then plated in 96-wellFlexible Plates (FALCON) at a cell density of 1×10⁵ cells/well. Aftercentrifugation at 700×g and 4° C. for two minutes (TOMY MultipurposeRefrigerated Centrifuge LX120), the supernatant was discarded. 5 μl ofFc Receptor Block was added to each well. After the plates were allowedto stand for five minutes, 50 μl of the hybridoma (1H12) supernatant wasadded to each well. The plates were allowed to stand at room temperaturefor 30 minutes. Isotypic control IgG2a (Immunotech) diluted ten timeswith PBS containing 2 mM EDTA and 0.5% BSA was used as a negativecontrol. After the antibody reaction, 100 μl of PBS containing 2 mM EDTAand 0.5% BSA was added to each well. The plates were centrifuged at700×g and 4° C. for two minutes, and the supernatant was discarded;another cycle of this treatment was performed for washing. Afterwashing, 40 μl of Goat F(ab′)2 Fragment Mouse IgG (H+L)-PE (Immunotech)diluted 200 times with PBS containing 2 mM EDTA and 0.5% BSA was addedto cells in each well, and the plates were allowed to stand at roomtemperature for 30 minutes. After secondary antibody reaction, theplates were washed three times. The cells were suspended in 1 ml of PBScontaining 2 mM EDTA and 0.5% BSA and were analyzed using a flowcytometer (Cytomics FC500 Beckman counter). The results are shown inFIG. 4 a.

3. Staining of PBMCs with an Anti-HIDE1 Antibody

The reactivity of an anti-HIDE1 antibody to peripheral blood mononuclearcell (PBMC) fraction was investigated to study human HIDE1 expression inblood cells. First, PBMCs were isolated from human peripheral bloodusing HISTOPAQUE-1077 (Sigma). The PBMCs were treated by the same FCMprocedure as described above in Section 2. More specifically, using flowcytometry the PBMCs were divided by two-dimensional fractionation interms of cell size and light scattering into three cellpopulations—lymphocytes, monocytes, and granulocytes—and then simplestained with an anti-HIDE1 antibody. As a result, it was found thatHIDE1 was expressed mainly in monocytes, but not in lymphocytes. Someweak expression was also observed in granulocytes, but the staining wassignificantly weaker than in monocytes (FIG. 5). This result stronglysuggests the possibility that HIDE1 can be used as a novel monocytemarker.

4. Double Staining of PBMCs Using an Anti-HIDE1 Antibody and AnotherDifferentiation Marker

PBMCs were suspended in PBS containing 2 mM EDTA and 0.5% BSA, andplated in each well of 96-well Flexible Plates (FALCON) at a celldensity of 1×10⁵ cells/well. After centrifugation at 700×g and 4° C. fortwo minutes (TOMY Multipurpose Refrigerated Centrifuge LX120), thesupernatant was discarded. 5 μl of Fc Receptor Block was added to eachwell. After the plates were allowed to stand for five minutes, 50 μl ofsupernatant of hybridoma (1H12) was added to each well. The plates wereallowed to stand at room temperature for 30 minutes. Isotypic controlIgG2a (Immunotech) diluted ten times with PBS containing 2 mM EDTA and0.5% BSA was used as a negative control.

After the antibody reaction, 100 μl of PBS containing 2 mM EDTA and 0.5%BSA was added to each well. The plates were centrifuged at 700×g and 4°C. for two minutes and the supernatant was discarded; another cycle ofthis treatment was then performed for washing. After washing, 40 μl ofGoat F(ab′)2 Fragment Mouse IgG (H+L)-FITC (Immunotech) diluted 200times with PBS containing 2 mM EDTA and 0.5% BSA was added to the cellsin each well. The plates were allowed to stand at room temperature for30 minutes. After the secondary antibody reaction, the cells were washedthree times. After washing, 50 μl of PE-labeled antibody diluted tentimes with PBS containing 2 mM EDTA and 0.5% BSA was added to the cellsin each well. The plates were allowed to stand for 30 minutes at roomtemperature. The antibodies used were CD11b, CD14, CD33, and Isotypiccontrol IgG1 (Immunotech). The cells were washed three times, andtransferred into FCM tubes. Each volume was adjusted to 1 ml with PBS.The samples were assayed using a flow cytometer.

To more closely investigate HIDE1 expression in monocytes, doublestaining was carried out using CD14, a monocyte marker (ref.2), incombination with an anti-HIDE1 antibody (FIG. 6). HIDE1 was found to beexpressed in 99% of CD14-positive cells (FIGS. 6 b and 6 c). SinceCD14-negative, HIDE1-positive cells account for 2% of PBMCs (FIG. 6 d),the anti-HIDE1 antibody could possibly detect CD14-negative monocytes.Specifically, the anti-HIDE1 antibody is expected to be able to stain abroader range of monocytes than CD14.

Double staining with the anti-HIDE1 antibody and CD11b (ref.3), anothermonocyte marker, was conducted (FIG. 7), and the results showed thatalmost all cells in the strongly CD11b-positive cell population werestained with HIDE1 (FIG. 7 b). The weakly CD11b-positive cell populationwas divided into HIDE1-negative and HIDE1-positive populations. Theweakly CD11b-positive, HIDE1-positive cell population mainly comprisedmonocytes (FIG. 7 c), while the weakly CD11b-positive, HIDE1-negativecell population was a lymphocyte population (FIG. 7 d). This resultsuggests that HIDE1 can stain monocytes more specifically than CD11b.

Double staining was carried out using the anti-HIDE1 antibody incombination with CD33 (ref.4), another monocyte marker (FIG. 8). It wasfound that CD33-positive cells account for 98% of HIDE1-positive cellsand HIDE1-positive cells account for 94% of CD33-positive cells (FIG. 8b). These findings show that HIDE1 is an entirely new monocyte marker.

5. Staining of Human Cultured Cells with an Anti-HIDE1 Antibody

Human cultured cells were stained with an anti-HIDE1 antibody using thesame FCM procedure as described in Section 2. The human cultured celllines used are THP-1, HL60, and U937. As a result HIDE1 was found to beexpressed in human monocytic cultured cell lines such as HL60, U937, andTHP-1 (FIG. 9). Thus, functional analyses of HIDE1 can be carried outusing these human monocytic cultured cell lines.

The expression of mRNA was analyzed, and in mice HIDE1 was found to beexpressed in dendritic cells derived from the spleen. Further, HIDE1gene was also detectable in human spleen cDNA libraries, suggesting thepossibility that HIDE1 is expressed in dendritic cells of human spleens(data not shown). However, HIDE1 was not expressed in dendritic cellsderived and differentiated from human peripheral blood monocytes usingGM-CSF and IL-4 (data not shown).

When THP-1, a monocytic cultured cell line, was differentiated intomacrophage-like cell using phorbol ester, a differentiation-inducingfactor, the expression level of HIDE1 was markedly reduced (FIG. 9 d).These results strongly suggest that HIDE1 is expressed specifically inmonocytes, and reinforce the fact that HIDE1 can be used as a monocytemarker. These results suggest the possibility that human HIDE1 isinvolved in the differentiation and/or growth of monocytes, and furtherthat human HIDE1 may also be involved in myelocytic leukemia. The invivo functions of HIDE1 can be clarified when ligands for HIDE1 areidentified in the future.

6. Western Blotting

Samples of hHIDE1-expressing 293T (H) and 293T (C) expressing anotherMyc tag fusion protein as a control were prepared in SDS-PAGE buffer,and electrophoresed using a 12.5% SDS-PAGE gel. Proteins fractionated bySDS-PAGE were transferred onto PVDF membrane (Millipore), and the blotmembrane was treated with Block Ace (Dainippon Pharmaceutical Co. Ltd.)for two hours for blocking. Then, the blot membrane was incubated withhybridoma supernatant (3F12) at room temperature for one hour. Toconfirm HIDE1 expression in 293T, the blot membrane was incubated withanti-Myc-tag monoclonal antibody (MBL) 6000 times diluted with BlockAce.

The membrane was next washed with PBS containing 0.5% Tween20, and thenincubated with anti-mouse IgG-POD conjugate (MBL) 3000 times dilutedwith Blue Buffer (20 mM HEPES, 1% BSA, 135 mM NaCl, 0.1% p-hydroxyphenylacetic acid, 0.15% cathonCG, and 10 μg/ml bromophenol blue) at roomtemperature for one hour. After secondary antibody reaction, themembrane was washed and bands of interest were detected bychemiluminescence using SuperSignal (Pierce). The molecular weight ofhHIDE1, estimated from the amino acid sequence, is 19 kDa. However, theextracellular domain of hHIDE1 is glycosylated at four sites, and as aresult the detected bands had shifted to positions at about 23 and 28kDa (FIG. 4 b).

7. Immunoprecipitation

0.5 mg of biotin (Pierce) was added to each of the cell suspensions (1ml) of 293T (H) expressing hHIDE1, and 293T (C) expressing anotherprotein (2×10⁷ cells /ml). The mixtures were stirred by inverting at 4°C. for one hour. After centrifugation at 4° C. and 200×g for fiveminutes the precipitates were washed four times with PBS. After washing,1 ml of Lysis buffer (10 mM Tris-HCl (pH 7.5), 1% NP-40, 150 mM NaCl,×200 Protease inhibitor cocktail (Sigma)) was added to the precipitates.Each sample was mixed by inverting every five minutes for 30 minutes onice. Then, the samples were centrifuged at 20,400×g for 15 minutes. Theresulting supernatants were collected, and combined with 50 μl ofrProteinA Sepharose FastFlow (Amersham Biosciences). The mixtures werestirred by inverting at 4° C. for 30 minutes, and then centrifuged at 4°C. and 600×g for one minute. The resulting supernatants were collected.The stirring was repeated once more and the supernatants were collected.

Each supernatant was mixed with anti-HIDE1 antibody-immobilizedSepharose (prepared by combining 1 ml of supernatant of theantibody-producing hybridoma (3H3) with 50 μl of rProteinA SepharoseFastFlow, and rotating at 4° C. for one hour). The resulting mixture wasstirred by inverting at 4° C. for one hour. After washing five times,the pellets were dissolved in SDS-PAGE buffer. The samples prepared wereelectrophoresed using a 12.5% SDS-PAGE gel. The proteins fractionated bythe same procedure as in Section 6 were transferred onto a PVDFmembrane. After the membrane was incubated with avidin-POD conjugate(MBL) diluted 10,000 times with blue buffer, bands of interest weredetected by chemiluminescence using SuperSignal. The results are shownin FIG. 4 c.

8. Double Staining of PBMCs Using an Anti-hHIDE1 Antibody and BDCA3, aMarker for Bone Marrow-Derived Dendritic Cells

PBMCs were collected from healthy donors and suspended in PBS containing2 mM EDTA and 0.5% BSA. The cells were plated in 96-well Flexible Plates(FALCON) at a cell density of 1×10⁵ cells/well. After centrifugation at700×g and 4° C. for two minutes (TOMY Multipurpose RefrigeratedCentrifuge LX120), the supernatant was discarded. 10 μl of Fc ReceptorBlock (1 mg/ml human IgG; Sigma) was added to each well. The plates wereallowed to stand at room temperature for ten minutes. 50 μl of BDCA3-PEantibody (Miltenyi Biotec) diluted ten times with PBS containing 2 mMEDTA and 0.5% BSA was added to each well, and the plates were allowed tostand at 4° C. for one hour. Isotypic control IgG1-PE (Immunotech)diluted ten times with PBS containing 2 mM EDTA and 0.5% BSA was used asa negative control. After the antibody reaction, 100 μl of PBScontaining 2 mM EDTA and 0.5% BSA was added to each well. The plateswere centrifuged at 700×g and 4° C. for two minutes and the supernatantswere discarded; another cycle of this treatment was performed forwashing. After washing, 50 μl of purified anti-hHIDE1 antibody (1H12)labeled with FITC was added at a concentration of 2 μg/ml to the cells.The resulting mixtures were allowed to stand at 4° C. for one hour.Isotypic control IgG1-FITC (Immunotech) diluted ten times with PBScontaining 2 mM EDTA and 0.5% BSA was used as a negative control. Afterwashing three times, each sample was transferred into a FCM tube, andthe volume was adjusted to 1 ml with PBS. The samples were assayed usinga flow cytometer. The results are shown in FIG. 10.

As a result, it was found that most BDCA3-positive cells were HIDE1-positive (enclosed by the broken line in FIG. 10 b). These results showthat HIDE1 is a marker for bone marrow-derived dendritic cells as wellas a monocyte marker, suggesting the possibility that monocytes and bonemarrow-derived dendritic cells can be collected simultaneously fromperipheral blood by using antibodies against HIDE1.

9. Preparation of Anti-Mouse HIDE1 Antibody

Mouse HIDE1 gene was amplified by PCR from a cDNA library of culturedcell DC2.4. The PCR product was cloned into pcDNA3.1 (Invitrogen), anexpression vector for cultured animal cells (pcDNA-mHIDE1). The vectorwas designed so that the C terminus of mouse HIDE1 (mHIDE1) was fusedwith Myc tag. pcDNA-mHIDE1 was transiently introduced into L cell, amouse cultured cell line, using Lipofectamine 2000 (Invitrogen). Wisterrats were immunized using the obtained mHIDE1-expressing cells as anantigen. Lymphocytes collected from lymph nodes of the immunized ratswere fused with Myeloma P3U1. After ten days, the supernatant of eachclone was sampled. Cell supernatants that reacted with the mHIDE1-expressing cell line were selected by flow cytometry and hybridomasproducing anti-mHIDE1 antibodies were thus obtained. The flow cytometryis described in detail below.

10. Flow Cytometry (FCM)

mHIDE1-expressing 293T and wild type 293T, or mHIDE1-expressing L-cellsand wild type L-cells were each suspended in PBS containing 2 mM EDTAand 0.5% BSA, and plated in 96-well Flexible Plats (FALCON) at a celldensity of 1×10⁵ cells/well. After centrifugation at 700×g and 4° C. fortwo minutes (TOMY Multipurpose Refrigerated Centrifuge LX120), thesupernatants were discarded. 50 μl of the hybridoma supernatant wasadded to each well. The plates were allowed to stand at room temperaturefor 30 minutes. Isotypic controls, IgG1, 2a, and 2b (MBL), 100 timesdiluted with PBS containing 2 mM EDTA and 0.5% BSA, were used asnegative controls.

After the antibody reaction, 100 μl of PBS containing 2 mM EDTA and 0.5%BSA was added to each well. The plates were centrifuged at 700×g and 4°C. for two minutes, and the supernatant was discarded; another cycle ofthis treatment was performed for washing. After washing, 40 μl of GoatF(ab′)2 Fragment Rat IgG (H+L)-PE (Immunotech) diluted 200 times withPBS containing 2 mM EDTA and 0.5% BSA was added to each well, and theplates were allowed to stand at room temperature for 30 minutes. Afterthe secondary antibody reaction, the plates were washed three times. Thecells were suspended in 1 ml of PBS containing 2 mM EDTA and 0.5% BSA,and analyzed using a flow cytometer (Cytomics FC500 Beckman counter).

11. Double Staining of PBMCs Using an Anti-mHIDE1 Antibody and aDifferentiation Marker

To test the reactivity of an anti-mHIDE1 antibody to peripheral bloodmononuclear cell (PBMC) fraction, PBMCs were isolated from theperipheral blood of c57BL6 mice using Lmpholyte-Mammal (CEDARLANE).PBMCs suspended in PBS containing 2 mM EDTA and 0.5% BSA were plated in96-well Flexible Plats (FALCON) at a cell density of 1×10⁵ cells/well.After centrifugation at 700×g and 4° C. for two minutes (TOMYMultipurpose Refrigerated Centrifuge LX120), the supernatants werediscarded. 50 μl of 100 times diluted Fc Receptor Block (CD16/32antibody; Pharmingen) was added to each well. The plates were allowed tostand at room temperature for 10 minutes. 50 μl aliquots of PE-labeledantibody diluted 100 times with PBS containing 2 mM EDTA and 0.5% BSAwere added, and the plates were allowed to stand at 4° C. for one hour.The antibodies used were CD11b, CD 11c, and Isotypic controls IgG2a and2b (Pharmingen).

After the antibody reaction, 100 μl of PBS containing 2 mM EDTA and 0.5%BSA was added to each well. The plates were centrifuged at 700×g and 4°C. for two minutes, and the supernatants were discarded; another cycleof this treatment was performed for washing. After washing, 50 μl of apurified anti-mHIDE1 antibody (SF8) labeled with FITC was added to thecells at a concentration of 2 μg/ml, and the resulting mixtures wereallowed to stand at 4° C. for one hour. Isotypic control IgG2b-FITC(Pharmingen), diluted 100 times with PBS containing 2 mM EDTA and 0.5%BSA, was used as a negative control. The cells were washed three times,and transferred into FCM tubes. The volume was adjusted to 1 ml withPBS. The samples were assayed using a flow cytometer. The results areshown in FIGS. 11 a and 11 b, and FIGS. 12 a and 12 b.

12. Staining of Mouse Spleen Cells with an Anti-mHIDE1 Antibody

To test the reactivity of an anti-mHIDE1 antibody to spleen cells,spleen cells were isolated from the spleens of c57BL6 mice. PBMCs weretreated by the same procedure as in Section 10. The results are shown inFIGS. 11 c and 11 d, and FIGS. 12 c and 12 d.

Mouse peripheral mononuclear blood (PBMC) and mouse spleen cells weredouble-stained with an anti-mHIDE1 antibody (5F8) and a dendritic cellmarker (CD11c). The results showed that a part of the CD11c-positivecells were HIDE1 -positive (enclosed by the broken line in FIGS. 12 band 12 d). CD11c is also expressed in NK cells. It was confirmed thatHIDE1 was not expressed in NK cells (data not shown).

The results described above show that like human HIDE1, mouse HIDE1 isexpressed in peripheral blood monocytes (FIG. 11) and dendritic cells(FIG. 12) as well as in splenic monocytes and dendritic cells (FIGS. 12and 13). Since human HIDE1 distribution was confirmed to correlate withmouse HIDE1 distribution, it is thought that the in vivo functions ofHIDE1 can be analyzed using mice in the future.

INDUSTRIAL APPLICABILITY

The present invention provided novel monocyte markers. Monocytes arecells which migrate in blood and which have phagocytotic activity,belonging to the group of mononuclear phagocytes. Once differentiated,monocytes remain in the bone marrow for only a short time beforeentering the circulation system, where they stay for several days.Monocytes then infiltrate tissues and body cavities, and differentiateinto macrophages and dendritic cells. Monocytes are known to increase inthe circulatory system during inflammatory responses, and thus monocytedetection is thought to be useful to detect inflammatory responses.Meanwhile, an increase in monocytes after organ transplantation suggeststhe possibility of tissue or graft rejection, and thus the monocytedetection of the present invention is useful for diagnoses after organtransplantation.

Since HIDE1 is expressed specifically in peripheral blood monocytes,HIDE1-positive monocytes can be collected from peripheral blood using acell sorter, magnet, or such. Monocytes are precursor cells of dendriticcells and macrophages, and dendritic cells and macrophages can beprepared from monocytes in vitro. Dendritic cells derived fromCD14-positive monocytes are used in cell immunotherapy (Pickl et al., J.Immunol. (1996) 157: 2850-9; Jefford et al., Blood (2003) 102: 1753).Monocytes selected using HIDE1 as a marker are also expected to beapplicable to cell immunotherapy. Since HIDE1 is not expressed inlymphocytes it can be used as a tool to enrich lymphocytes by removingHIDE1-positive cells from peripheral blood using an anti-HIDE1 antibody.

Methods for treating tumors and viral infections using activatedlymphocytes have been proposed. Thus, cells that can be used to treatand prevent cancers, viral infections, spinal cord damage, and variousother diseases for which the administration of monocytes, macrophages,dendritic cells, or lymphocytes is effective, can be preparedefficiently by using an antibody that recognizes an HIDE1 marker of thepresent invention. Human HIDE1 maybe involved in the differentiationand/or growth of monocytes, and may also be involved in myelocyticleukemias. When ligands for HIDE1 are identified, the in vivo functionsof HIDE1 are expected to be further clarified.

1. An antibody for detecting a monocyte marker, which binds to a proteinor a polypeptide selected from the group of: (1) an HIDE1 protein; (2) aprotein encoded by a nucleotide sequence that hybridizes to acomplementary sequence of an HIDE1 gene under stringent conditions; and(3) a polypeptide fragment with at least eight amino acid residues,wherein the fragment is derived from the protein of the above (1) or(2).
 2. A method for detecting a monocyte, which comprises the steps of:(1) contacting the antibody of claim 1 with a blood cell samplepredicted to comprise a monocyte, and (2) detecting a blood cell thathas bound to the antibody in step (1).
 3. A method for isolating amonocyte, which comprises the steps of: (1) contacting the antibody ofclaim 1 with a blood cell sample predicted to comprise a monocyte, and(2) collecting a blood cell that has bound to the antibody in step (1).4. The method of claim 2 or 3, wherein the blood cell sample isperipheral blood, cord blood, or bone marrow.
 5. A kit for detectingand/or isolating a monocyte, which comprises the antibody of claim
 1. 6.A method for inducing a dendritic cell from a monocyte in vitro, whichcomprises the steps of: (1) culturing a monocyte in the presence of adifferentiation inducing factor for a dendritic cell; and (2) contactinga cell cultured in step (1) with the antibody of claim 1, detectingHIDE1 expression, and judging that the differentiation of a monocyteinto a dendritic cell is induced when the expression level of HIDE1 isreduced.
 7. The method of claim 6, wherein the differentiation inducingfactor for a dendritic cell is a combination of GM-CSF and IL-4.
 8. Amethod for inducing a macrophage from a monocyte in vitro, whichcomprises the steps of: (1) culturing a monocyte in the presence of adifferentiation inducing factor for a macrophage; and (2) contacting acell cultured in step (1) with the antibody of claim 1, detecting HIDE1expression, and judging that the differentiation of a monocyte into amacrophage-like cell is induced when the expression level of HIDE1 isreduced.
 9. The method of claim 8, wherein the differentiation inducingfactor for a macrophage is phorbol ester.
 10. A method for obtaining adendritic cell, which comprises the steps of: (1) contacting a sample ofcollected blood cells with the antibody of claim 1; (2) collecting ablood cell that has bound to the antibody in step (1); (3) culturing theblood cell collected in step (2) in the presence of a differentiationinducing factor for a dendritic cell; (4) contacting the cell culturedin step (3) with the antibody of claim 1, detecting HIDE1 expression,and judging that a monocyte is differentiated into a dendritic cell whenthe expression level of HIDE1 is reduced; and (5) isolating as adendritic cell the cell judged to be differentiated in step (4).
 11. Themethod of claim 10, which further comprises the step of allowing theisolated dendritic cell to ingest an antigen.
 12. The method of claim10, wherein the isolated dendritic cell is used to prevent and/or treata tumor.
 13. The method of claim 12, which further comprises the step ofallowing the isolated dendritic cell to ingest a tumor-specific antigen.14. The method of claim 10, wherein the isolated dendritic cell is usedto prevent and/or treat an autoimmune disease, or to relieve rejectionafter an organ transplantation.
 15. A method for obtaining a macrophage,which comprises the steps of: (1) contacting a sample of collected bloodcells with the antibody of claim 1; (2) collecting a blood cell that hasbound to the antibody in step(1); (3) culturing the blood cell collectedin step (2) in the presence of a differentiation inducing factor for amacrophage; (4) contacting the cell cultured in step (3) with theantibody of claim 1, detecting HIDE1 expression, and judging that amonocyte is differentiated into a macrophage-like cell when theexpression level of HIDE1 is reduced; and (5) isolating as a macrophagethe cell judged to be differentiated in step (4).
 16. The method ofclaim 15, which further comprises the step of activating the isolatedcell.
 17. The method of claim 15 or 16, wherein the isolated macrophageis used to treat a spinal cord damage, and/or to treat and/or prevent atumor, infectious disease, autoimmune disease, or immunodeficiencydisease.
 18. A method for collecting a lymphocyte, which comprises thesteps of: (1) contacting the antibody of claim 1 with a blood cellsample predicted to comprise a lymphocyte; and (2) collecting a bloodcell that did not bind to the antibody in step (1).
 19. A method forobtaining an activated lymphocyte, which comprises the steps of: (1)contacting the antibody of claim 1 with a blood cell sample predicted tocomprise a lymphocyte; (2) collecting as a lymphocyte a blood cell thatis not bound to the antibody; (3) culturing the lymphocyte collected instep (2); and (4) activating the lymphocyte cultured in step (3) andcollecting the activated lymphocyte.
 20. The method of claim 18 or 19,wherein the blood cell sample is peripheral blood, cord blood, or bonemarrow.
 21. The method of claim 19, wherein the activated lymphocyte isused to prevent and/or treat a tumor or infectious disease.